Disclosure of Invention
The invention aims to provide a recombinant human thymosin beta 4 (T beta 4) microneedle patch, a preparation method and application thereof in wound healing, the recombinant human thymosin beta 4 microneedle patch prepared by the invention has high drug-loading capacity, good molding and high mechanical strength, can rapidly and continuously release most of drugs in a short time, is beneficial to transdermal penetration of T beta 4 drugs, and has good promotion effect on wound healing.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a recombinant human thymosin beta 4 microneedle patch, which comprises the following steps:
Mixing recombinant human thymosin beta 4, a first auxiliary material and water to obtain a microneedle solution, wherein the first auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;
mixing a second auxiliary material with water to obtain a supporting layer solution, wherein the second auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;
Adding the microneedle solution into a microneedle mould, and then carrying out exhaust treatment, wherein after the exhaust treatment, the supporting layer solution is added into the microneedle mould to obtain a formed body;
And drying the formed body, and demolding to obtain the recombinant human thymosin beta 4 microneedle patch, wherein the drying temperature is lower than 40 ℃.
Preferably, the drying temperature is less than or equal to 35 ℃ and the drying time is more than or equal to 36 hours.
Preferably, the mass concentration of the recombinant human thymosin beta 4 in the microneedle solution is 10-400 mg/mL.
Preferably, the first auxiliary material is chondroitin sulfate and sucrose, wherein the content of the chondroitin sulfate in the microneedle solution is 10-100 w/v%, and the content of the sucrose is 0.1-10 w/v%.
Preferably, the first auxiliary material comprises hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol, wherein the content of the hydroxypropyl-beta-cyclodextrin in the microneedle solution is 10-100 w/v%, the content of the tween 80 is 0.1-10 w/v%, the content of the trehalose is 0.1-30 w/v%, and the content of the polyvinyl alcohol is 10-100 w/v%.
Preferably, the second auxiliary material is chondroitin sulfate and sucrose, wherein the content of the chondroitin sulfate in the supporting layer solution is 10-100 w/v%, and the content of the sucrose is 0.1-10 w/v%.
Preferably, the second auxiliary material comprises hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol, wherein the content of the hydroxypropyl-beta-cyclodextrin in the supporting layer solution is 10-100 w/v%, the content of the tween 80 in the supporting layer solution is 0.1-10 w/v%, the content of the trehalose in the supporting layer solution is 0.1-30 w/v%, and the content of the polyvinyl alcohol in the supporting layer solution is 10-100 w/v%.
Preferably, the microneedle mould comprises a supporting layer structure and microneedle structures arranged on the surface of the supporting layer structure, wherein the microneedle structures are arranged on the surface of the supporting layer structure in an array mode, the microneedle structures are hollow cones, the diameter of the bottoms of the microneedle structures is 100-500 mu m, the height of the microneedle structures is 300-1000 mu m, and the top intervals of any 2 adjacent microneedle structures are 500-1200 mu m.
The invention provides a recombinant human thymosin beta 4 microneedle patch prepared by the preparation method according to the technical scheme, which comprises a supporting layer and microneedles arranged on the surface of the supporting layer in an array manner, wherein the microneedles comprise recombinant human thymosin beta 4 and first auxiliary materials, the first auxiliary materials comprise at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose, the supporting layer comprises second auxiliary materials, and the second auxiliary materials comprise at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose.
The invention provides an application of the recombinant human thymosin beta 4 microneedle patch in manufacturing medical devices for wound healing.
The invention provides a preparation method of a recombinant human thymosin beta 4 microneedle patch, which comprises the steps of mixing recombinant human thymosin beta 4, a first auxiliary material and water to obtain a microneedle solution, wherein the first auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose, mixing a second auxiliary material and water to obtain a supporting layer solution, the second auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose, adding the microneedle solution into a microneedle mould, then carrying out exhaust treatment, adding the supporting layer solution into the microneedle mould after the exhaust treatment to obtain a formed body, drying the formed body, and demolding to obtain the recombinant human thymosin beta 4 microneedle patch, wherein the drying temperature is lower than 40 ℃. The invention can effectively avoid the problem that the recombinant human thymosin beta 4 is likely to lose activity or lose the drug effect caused by structural change under the high temperature condition by reasonably setting the types of auxiliary materials in the microneedle solution and the supporting layer solution and adopting the low temperature condition with the temperature of less than 40 ℃ for drying, and the prepared recombinant human thymosin beta 4 microneedle patch has high drug loading capacity, and meanwhile, the obtained recombinant human thymosin beta 4 microneedle patch has good molding and high mechanical strength, can rapidly and continuously release most of drugs in a short time, and is beneficial to the percutaneous permeation of T beta 4 drugs. The results of the examples show that the recombinant human thymosin beta 4 microneedle patch prepared by the invention has good molding, good appearance of needle shape, no empty needle phenomenon, no sharp needle tip or breakage phenomenon, and sharp needle tip, can easily penetrate through aluminum foil, has enough mechanical strength, each single needle can bear 0.47N force, the force required for penetrating through skin is usually less than 0.1N per needle, so that the microneedles can easily penetrate through skin and have good mechanical properties, the drug loading amount of the recombinant human thymosin beta 4 microneedle patch prepared by the invention is 200-1000 mug of drug loading amount of each microneedle, and the recombinant human thymosin beta 4 microneedle patch prepared by the invention can rapidly and continuously release most of drugs within 30min, is favorable for transdermal penetration of drugs, has good safety, and has good effect of promoting wound healing.
Detailed Description
The invention provides a preparation method of a recombinant human thymosin beta 4 microneedle patch, which comprises the following steps:
Mixing recombinant human thymosin beta 4, a first auxiliary material and water to obtain a microneedle solution, wherein the first auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;
mixing a second auxiliary material with water to obtain a supporting layer solution, wherein the second auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;
Adding the microneedle solution into a microneedle mould, and then carrying out exhaust treatment, wherein after the exhaust treatment, the supporting layer solution is added into the microneedle mould to obtain a formed body;
And drying the formed body, and demolding to obtain the recombinant human thymosin beta 4 microneedle patch, wherein the drying temperature is lower than 40 ℃.
In the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
The invention mixes recombinant human thymosin beta 4, a first auxiliary material and water to obtain a microneedle solution, wherein the first auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose. In the present invention, the first auxiliary material is preferably chondroitin sulfate and sucrose. Or the first auxiliary material is preferably hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol. The water is particularly preferably ultrapure water. The mass concentration of the recombinant human thymosin beta 4 in the microneedle solution is preferably 10-400 mg/mL, more preferably 10-150 mg/mL, and even more preferably 50-100 mg/mL. When the first auxiliary material is preferably chondroitin sulfate and sucrose, the content of the chondroitin sulfate in the microneedle solution is preferably 10-100 w/v%, more preferably 20-80 w/v%, and the content of the sucrose is preferably 0.1-10 w/v%, more preferably 1-8 w/v%. When the first auxiliary material is preferably hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol, the content of the hydroxypropyl-beta-cyclodextrin in the microneedle solution is preferably 10-100 w/v%, more preferably 20-80 w/v%, the content of tween 80 is preferably 0.1-10 w/v%, more preferably 1-8 w/v%, the content of trehalose is preferably 0.1-30 w/v%, more preferably 1-25 w/v%, and the content of polyvinyl alcohol is preferably 10-100 w/v%, more preferably 20-80 w/v%.
In the invention, the mixing of the recombinant human thymosin beta 4, the first auxiliary material and the water preferably comprises the steps of dissolving the recombinant human thymosin beta 4 in the water to obtain an aqueous solution of the recombinant human thymosin beta 4, and adding the first auxiliary material into the aqueous solution of the recombinant human thymosin beta 4.
The preparation method comprises the step of mixing a second auxiliary material with water to obtain a supporting layer solution, wherein the second auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose. In the present invention, the second auxiliary material is preferably chondroitin sulfate and sucrose. Or the second auxiliary material is preferably hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol. The water is particularly preferably ultrapure water. When the second auxiliary material is preferably chondroitin sulfate and sucrose, the content of the chondroitin sulfate in the supporting layer solution is preferably 10-100 w/v%, more preferably 20-80 w/v%, and the content of the sucrose is preferably 0.1-10 w/v%, more preferably 1-8 w/v%. When the second auxiliary material is preferably hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol, the content of the hydroxypropyl-beta-cyclodextrin in the supporting layer solution is preferably 10-100 w/v%, more preferably 20-80 w/v%, the content of tween 80 is preferably 0.1-10 w/v%, more preferably 1-8 w/v%, the content of trehalose is preferably 0.1-30 w/v%, more preferably 1-25 w/v%, and the content of polyvinyl alcohol is preferably 10-100 w/v%, more preferably 20-80 w/v%.
After the microneedle solution is obtained, the microneedle solution is added into a microneedle mould and then subjected to exhaust treatment. In the present invention, the material of the microneedle mould is preferably polydimethylsiloxane. The microneedle mould comprises a supporting layer structure and microneedle structures arranged on the surface of the supporting layer structure, wherein the microneedle structures are arranged on the surface of the supporting layer structure in an array mode, the shape of each microneedle structure is a hollow cone, the diameter of the bottom of each microneedle structure is preferably 100-500 microns, more preferably 200-400 microns, the height of each microneedle structure is preferably 300-1000 microns, more preferably 300-1000 microns, and the top distance of any adjacent 2 microneedle structures is preferably 500-1200 microns, more preferably 600-1000 microns. In the invention, the supporting layer structure of the microneedle grinding tool is square, and the long side of the square supporting layer structure is 10-50 mm. In a specific embodiment of the present invention, the size of the supporting layer structure of the microneedle grinder may be 10.5mm×10.5mm or 50mm×50mm, and the microneedle structures are preferably arranged in a 10×10 square array on the surface of the supporting layer structure.
In the invention, the exhaust treatment is preferably performed under vacuum conditions, and the vacuum degree is preferably-0.09 to-0.1 MPa. In a specific embodiment of the present invention, the evacuation treatment is preferably performed in a vacuum drying oven. The exhaust treatment is preferably carried out circularly, the single exhaust treatment step comprises the steps of vacuumizing the exhaust treatment environment to-0.09 to-0.1 MPa, maintaining for 1-5 min, and then carrying out the normal pressure of the deflation value, wherein the exhaust treatment is preferably carried out circularly for 1-5 times.
After the exhaust treatment and after the support layer solution is obtained, the support layer solution is added into the microneedle mould to obtain a molded body. The present invention preferably scrapes off excess microneedle solution located on the structural portion of the support layer prior to adding the support layer solution to the microneedle mould.
After the molded body is obtained, the molded body is dried, and the recombinant human thymosin beta 4 microneedle patch is obtained after demolding, wherein the drying temperature is less than 40 ℃, more preferably 2-30 ℃, and even more preferably 2-15 ℃. In the invention, the drying time is preferably not less than 36 hours, more preferably 36-48 hours. The drying is preferably carried out in a dryer.
The invention provides a recombinant human thymosin beta 4 microneedle patch prepared by the preparation method according to the technical scheme, which comprises a supporting layer and microneedles arranged on the surface of the supporting layer in an array manner, wherein the microneedles comprise recombinant human thymosin beta 4 and first auxiliary materials, the first auxiliary materials comprise at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose, the supporting layer comprises second auxiliary materials, and the second auxiliary materials comprise at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose.
In the present invention, the first auxiliary material is preferably chondroitin sulfate and sucrose. Or the first auxiliary material is preferably hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol.
In the present invention, the second auxiliary material is preferably chondroitin sulfate and sucrose. Or the second auxiliary material is preferably hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol.
In the invention, the active pharmaceutical ingredient of the recombinant human thymosin beta 4 microneedle patch is recombinant human thymosin beta 4. The recombinant human thymosin beta 4 microneedle patch is a soluble drug-loaded microneedle patch.
In the invention, the shape of the microneedle of the recombinant human thymosin beta 4 microneedle patch is preferably cone, the diameter of the bottom of the microneedle is preferably 100-500 μm, more preferably 200-400 μm, the height is preferably 300-1000 μm, more preferably 800-1000 μm, and the distance between the tips of any adjacent 2 microneedles is preferably 500-1200 μm, more preferably 600-1000 μm.
In the embodiment of the invention, the shape of the supporting layer of the recombinant human thymosin beta 4 microneedle patch is square, and the side length is 1-15 cm, preferably 1-5 cm. The size of the support layer of the recombinant human thymosin beta 4 microneedle patch is specifically 1cm×1cm or 5cm×5cm.
In the embodiment of the invention, the microneedles of the recombinant human thymosin beta 4 microneedle patch are arranged in a10×10-20×20 square array on the surface of the supporting layer.
The invention provides an application of the recombinant human thymosin beta 4 microneedle patch in manufacturing medical devices for wound healing. In the present invention, the medical device for wound healing is particularly preferably a microneedle patch.
The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Dissolving Tbeta 4 with ultrapure water to prepare Tbeta 4 solution, and adding chondroitin sulfate and sucrose to prepare the microneedle solution, wherein the mass concentration of the Tbeta 4 in the microneedle solution is 80mg/mL, the content of the chondroitin sulfate is 50% (w/v), and the content of the sucrose is 1% (w/v).
2. The supporting layer solution was prepared by adding chondroitin sulfate and sucrose to ultrapure water, wherein the content of chondroitin sulfate in the supporting layer solution was 50% (w/v), and the content of sucrose was 1% (w/v).
3. The method comprises the steps of adding a microneedle solution into a polydimethylsiloxane microneedle mould, wherein the microneedle mould comprises a supporting layer structure and microneedle structures arranged on the surface of the supporting layer structure, the size of the supporting layer structure is 1cm multiplied by 1cm or 5cm multiplied by 5cm, the microneedle structures are arranged in a 10 multiplied by 10 square array on the surface of the supporting layer structure, the microneedle structures are hollow cones, the diameter of the bottoms of the microneedle structures is 350 mu m, the height of the bottoms of the microneedle structures is 1000 mu m, and the top intervals of any two adjacent microneedle structures are 800 mu m. Then placing into a vacuum drying oven, vacuumizing to-0.1 MPa, keeping for 3min, then deflating, removing bubbles, vacuumizing again to-0.1 MPa, keeping for 2min, and then deflating.
4. The excess microneedle solution was scraped off and added to the support layer solution.
5. And (3) placing the microneedle mould containing the microneedle solution and the supporting layer solution in the step (4) in a dryer, and drying for 48 hours in an environment of 2-4 ℃ to obtain the Tbeta 4 drug-loaded microneedle patch.
Test example 1:T beta 4 drug-loaded microneedle patch morphology characterization
The morphology of the T beta 4 drug-loaded microneedle patch was observed by using a microscope, and the results are shown in fig. 1 and 2. From fig. 1 and fig. 2, it can be seen that the tβ4 drug-loaded microneedle patch prepared in example 1 has good molding, good appearance of needle shape, no empty needle phenomenon, no defect or fracture phenomenon at the tip, and sharp needle tip.
Test example 2:T beta 4 drug-loaded microneedle patch hardness investigation
The mechanical properties of the microneedles of the tβ4 drug-loaded microneedle patches were examined using an aluminum foil puncture test and an in vitro rat skin puncture test, respectively.
(1) Aluminium foil puncture test
When the aluminum foil is punctured, the micro needle is vertically punctured into the aluminum foil with a certain force and maintained for 2min. The microneedle was observed for penetration of the aluminum foil and evaluated for microneedle hardness. As a result, see fig. 3, and as a result of fig. 3, the micro needle of the tβ4 drug-loaded micro needle patch prepared in example 1 can easily penetrate through the aluminum foil, which indicates that the tβ4 drug-loaded micro needle patch prepared in example 1 has sufficient mechanical strength.
(2) Skin penetration test of isolated rats
Treatment of ex vivo skin
Healthy SD rats are taken, cervical vertebra is removed, the abdominal hair is carefully removed, the skin is removed, adipose tissues and fascia are peeled off, the healthy SD rats are repeatedly washed by normal saline, and then are sucked to dryness by filter paper, are wrapped by tinfoil paper, and are then put into a refrigerator at-80 ℃ for preservation. When in use, the skin is soaked in physiological saline for 30min.
Skin puncture of isolated rat
The microneedles of the tβ4 drug-loaded microneedle patch prepared in example 1 were vertically penetrated into the isolated skin of healthy intact rats with a certain force, the microneedles were taken out after 10min, the skin was immediately stained with 0.4% trypan blue solution, the superfluous stain on the skin surface was removed with isopropyl alcohol after 15min, and the skin surface was cleaned with physiological saline to observe the condition of the stained pinholes. The well visible pinholes were visible on the rat skin after trypan blue staining, see figure 4. The results of fig. 4 show that the tβ4 drug-loaded microneedle patch prepared in example 1 has sufficient mechanical strength to penetrate the skin ex vivo and meet the test requirements.
Test example 3:T beta 4 drug-loaded microneedle mechanical property test
The microneedles need to have some mechanical strength to ensure that they can puncture the skin barrier without breaking or bending. To test the mechanical strength of the microneedles, the mechanical strength of the tβ4 drug-loaded microneedle patches prepared in example 1 was evaluated using a texture analyzer. The test speed is set to be 0.05mm/s, and the mechanical property of the microneedle is evaluated by comparing the relation between the force born by the tip of the microneedle in the process of pressing down the probe and the displacement of the probe.
As a result, as shown in FIG. 5, when the force reaches 47N, the force-displacement curve suddenly drops, indicating that the needle tip breaks, and each single needle can be subjected to a force of 0.47N, and the force required for penetrating the skin is usually less than 0.1N/needle, so that the micro needle can easily penetrate the skin, and has good mechanical properties.
Test example 4:T beta 4 drug-loaded microneedle patch drug-containing amount test
Microneedle drug loading assay
The tβ4 drug-loaded microneedle patch prepared in example 1 was placed in a release medium using pH 7.4PBS as the release medium, and sampled by ultrasound for 3min, and the above operations were performed in 3 groups in parallel. Liquid chromatography is used for detection.
The average value of the detection results is 248.15 mug of drug loading rate of each microneedle.
Test example 5:T beta 4 drug-loaded microneedle in vitro release experiment
Adding PBS (phosphate buffer solution) with pH of 7.4 as a release medium into a small beaker, placing the T beta 4 drug-loaded microneedle patch prepared in the embodiment 1 into the release medium, placing the small beaker into a constant temperature shaking table, setting the temperature to 37 ℃, and sampling 400 mu L for 15s, 30s, 1min, 2min, 5min, 10min, 20min, 30min, 45min, 60min, 120min, 180min, 240min, 300min, 360min and 420min respectively, and simultaneously rapidly supplementing the release medium with the same volume. The above experiment was repeated 3 times (n=3), the content of the drug in the sample of each period was measured by high performance liquid chromatography, the chromatographic detection conditions were the same as those of test example 3, and the Cumulative release amount (cumulat ReleaseAmount, CRA) and the Cumulative release degree (cumulat RELEASE RATE, CRR) of the drug were calculated. The formula of the CRA is shown in formula 2:
In the formula 2, V is the volume of the release medium, vi is the volume of each sampling, and Ci-1 are the drug concentrations in the release medium at the ith and ith-1 th sampling respectively.
Cumulative release = cumulative release/total drug content x 100%.
TABLE 1 drug Release results for T beta 4 drug-loaded microneedle patch prepared in EXAMPLE 1
| Time/min | Degree of Release (%) |
| 0.25 | 36.54±8.91 |
| 0.5 | 56.62±1.36 |
| 1 | 62.10±6.36 |
| 2 | 69.07±13.74 |
| 5 | 70.45±20.03 |
| 10 | 70.70±17.04 |
| 20 | 77.83±10.86 |
| 30 | 86.67±4.42 |
| 45 | 99.70±0.23 |
| 60 | 93.92±0.87 |
| 120 | 91.71±3.38 |
| 180 | 89.69±0.46 |
| 240 | 86.02±4.45 |
| 300 | 84.12±2.70 |
| 360 | 85.75±3.45 |
| 420 | 86.09±1.61 |
The drug release profile of the tβ4 drug loaded microneedle patch prepared in example 1 is shown in fig. 6. As shown in fig. 6, the cumulative release degree of the drug-loaded microneedle is 62.10% at 1min, 86.67% at 30min and 99.70% at 45min, and the result proves that the tβ4 drug-loaded microneedle can rapidly and continuously release most of the drug within 45min, and the rapid release of the tβ4 microneedle is beneficial to the percutaneous permeation of the drug.
Test example 6:T beta 4 drug-loaded microneedle safety evaluation
Microneedles may break during use and remain in the skin for a long period of time, so the safety of the material is critical. The irritation test is to observe whether the blood vessel, muscle, skin, mucosa, etc. of animal is in contact with the test substance to cause local reaction such as red swelling, congestion, exudation, denaturation or necrosis.
SD rats were intraperitoneally injected with 150. Mu.L of 4% chloral hydrate and anesthetized, and abdominal hair was removed. The microneedle was attached to the abdominal skin of the rat for 10min, and then taken down to record the skin state, at this time, the skin state was recorded as 0min, and the skin recovery state was recorded as 2min, 4min, 6min, 8min, 10min, 30min, 1h, and 2h, respectively, and the skin recovery result is shown in fig. 7.
As shown in FIG. 7, the trace of the holes penetrated by the micro-needles is clear at 0min, and bleeding and redness are not caused. The holes on the skin are gradually blurred within 2min, the marks on the skin almost disappear after 10min, the skin returns to normal after 1h, and the marks are completely eliminated. The safety of the microneedles was found to be good by the extent of erythema and edema of the skin after application of the microneedles.
Test example 7:T beta 4 drug-loaded microneedle for wound healing study
(1) Mouse full cortex injury model establishment
After 20C 57 mice are selected and anesthetized by 4% chloral hydrate (100 mu L/10 g), the mice are fixed in a prone position, the hairs on the back of the mice are shaved off, the hairs are removed by using depilatory cream, after the operation part is sterilized, the whole skin is sheared off by tissues to prepare a circular wound surface (with the diameter of about 8 mm), a healing gasket is sewn, and the full-skin injury model of the mice is successfully established. FIG. 8 is a graph showing the results of a full-cortex injury model of mice.
(2) Grouping and processing of laboratory animals
The trauma modeling mice were randomly divided into 2 groups, the back wound of the experimental group mice was covered with Tss 4 drug-loaded microneedles, fixed with pressure-sensitive adhesive tape, and the blank control group was not covered with microneedles. Each group is divided into 10, and the microneedles are replaced every day.
(3) Wound surface observation and wound healing rate
The wounds were photographed and sampled (aligned with ruler) daily during the test period, the healing condition of the wounds was compared and recorded, the wound area of the mice in the specific period was calculated using Image J software, and the healing rate of the wounds was calculated. The calculation formula of the wound healing rate is shown in formula 3:
Healing rate = (day 0 wound area-determination of day wound area)/day 0 wound area x 100% 3
Fig. 9 is a diagram of wound healing. FIG. 10 is a graph of wound recovery rate
As seen from the animal test results (fig. 9 and 10), the tβ4 microneedle test group has a significant difference in wound healing rate from the 4 th day compared with the blank group, the microneedle group has almost healed up to 95.65% on the 12 th day of the test, the control group has only 86.44% of recovery rate, the microneedle group has basically recovered to normal on the 14 th day, and the control group is still in the healing process. It is shown that tβ4 micro-targets have a promoting effect on wound healing.
Example 2
1. Dissolving Tbeta 4 with ultrapure water to prepare Tbeta 4 solution, adding hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol to prepare the microneedle solution, wherein the mass concentration of the Tbeta 4 in the microneedle solution is 80mg/mL, the content of the hydroxypropyl-beta-cyclodextrin is 10% (w/v), the content of the Tween 80 is 0.1% (w/v), the content of the trehalose is 10% (w/v), and the content of the polyvinyl alcohol is 10% (w/v).
2. Adding hydroxypropyl-beta-cyclodextrin, tween 80, trehalose and polyvinyl alcohol into ultrapure water to prepare a supporting layer solution, wherein the content of the hydroxypropyl-beta-cyclodextrin in the supporting layer solution is 10% (w/v), the content of the Tween 80 is 0.1% (w/v), the content of the trehalose is 10% (w/v), and the content of the polyvinyl alcohol is 10% (w/v).
3. The method comprises the steps of adding a microneedle solution into a polydimethylsiloxane microneedle mould, wherein the microneedle mould comprises a supporting layer structure and microneedle structures arranged on the surface of the supporting layer structure, the size of the supporting layer structure is 10.5mm multiplied by 10.5mm, the microneedle structures are distributed in an 8 multiplied by 8 square array on the surface of the supporting layer structure, the microneedle structures are hollow cones, the diameter of the bottoms of the microneedle structures is 350 mu m, the height of the bottoms of the microneedle structures is 1000 mu m, and the top intervals of any 2 adjacent microneedle structures are 800 mu m. Then placing into a vacuum drying oven, vacuumizing to-0.1 MPa, keeping for 3min, then deflating, removing bubbles, vacuumizing again to-0.1 MPa, keeping for 2min, and then deflating.
4. The excess microneedle solution was scraped off and added to the support layer solution.
5. And (3) placing the microneedle mould containing the microneedle solution and the supporting layer solution in the step (4) in a dryer, and drying for 48 hours in an environment of 2-4 ℃ to obtain the Tbeta 4 drug-loaded microneedle patch.
The mechanical strength performance test of the T beta 4 drug-loaded microneedle patch prepared in example 2 was performed according to the method described in the test example 3:T beta 4 drug-loaded microneedle mechanical performance test, the test result is shown in fig. 11, the abscissa X in fig. 1 is displacement, the unit mm, the ordinate Y is force, and the unit N, and as can be seen from fig. 11, the average mechanical strength of 4 needles of the T beta 4 drug-loaded microneedle patch prepared in example 2 is 1.05+/-0.03N, which is greater than 0.2N, and meets the requirements.
The tβ4 drug-loaded microneedle patch prepared in example 2 was subjected to an in vitro release test according to the method described in the "test example 5:T β4 drug-loaded microneedle in vitro release test" above, and the release degree of the tβ4 drug-loaded microneedle patch prepared in example 2 is shown in table 2.
Table 2 release results of tβ4 drug loaded microneedle patch prepared in example 2
The drug release profile of the tβ4 drug loaded microneedle patch prepared in example 2 is shown in fig. 12. As shown in fig. 12, the tβ4 drug-loaded microneedle patch prepared in example 2 had similar properties in terms of wound healing as those of example 1.
Comparative example 1
The preparation process was essentially the same as in example 1, except that the drying process in step 5 was 40℃forced air drying oven drying for 24 hours.
Drug-loading measurement of T beta 4 drug-loading microneedle patch prepared in comparative example 1
5ML of PBS with pH=7.4 is used as a release medium, a mold with a needle tip part filled with the needle tip solution is placed in the release medium, ultrasonic is performed for 3min for sampling, 3 groups of the above operations are performed in parallel (n=3), and chromatographic detection conditions are the same as those of test example 4.
The areas of the three groups of parallel chromatographic peaks are 9.252, 11.877 and 10.876 respectively, and the three groups of parallel chromatographic peak areas are substituted into a linear standard curve equation 1 to obtain the drug loading of the microneedle prepared in comparative example 1, and the average drug loading value of each microneedle is 95.126 mug. As can be seen from the comparison of the drug loading of the Tss 4 drug-loaded microneedle patch prepared in example 1 obtained in test example 4, the microneedle patch prepared in comparative example 1 has a much lower drug content than the microneedle patch prepared in example 1 by the low-temperature drying method, compared with the microneedle patch prepared in example 1 by the low-temperature drying method.
The above examples show that the invention adopts chondroitin sulfate and sucrose as auxiliary materials of recombinant human thymosin beta 4, and simultaneously adopts low temperature condition with temperature less than or equal to 4 ℃ for drying, thereby effectively avoiding the problem that recombinant human thymosin beta 4 may lose activity or have structural change to cause the loss of drug effect under high temperature condition. The results of the examples show that the recombinant human thymosin beta 4 microneedle patch prepared by the invention has good molding, good appearance of needle shape, no empty needle phenomenon, no sharp needle tip or breakage phenomenon, and can easily penetrate through aluminum foil, has enough mechanical strength, each single needle can bear 0.47N force, the force required for penetrating through the skin is usually smaller than 0.1N per needle, so that the microneedles can easily penetrate through the skin, and have good mechanical properties, the drug loading amount of the recombinant human thymosin beta 4 microneedle patch prepared by the invention is 248.15 mu g per microneedle drug loading amount, the recombinant human thymosin beta 4 microneedle patch prepared by the invention can rapidly and continuously release most of drugs within 20min, the rapid release is favorable for transdermal penetration of the drugs, the recombinant human thymosin beta 4 microneedle patch prepared by the invention has good safety, and the recombinant human thymosin beta 4 microneedle patch prepared by the invention has good promotion effect on wound healing.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.